Project Summary Single-molecule approaches are revolutionizing biological inquiries by providing previously unattainable data on elementary biological processes. However, most single-molecule studies have been limited to single isolated molecules of protein, RNA, or DNA, yet we know that these molecules do not function in isolation in the cell. To better emulate in vivo conditions, we need to study more complex systems built of many components. The Johns Hopkins University has a large group (11 PIs) of NIH-funded investigators in the Schools of Medicine, Public Health, and Arts and Sciences that will utilize single-molecule measurement technologies to study a range of problems central to the NIH mission. As part of an established core facility, we request a Lumicks C-Trap that combines three state-of-the-art single-molecule measurement modalities into one instrument. It will be the first instrument in the mid-Atlantic area that has ultrahigh-resolution optical tweezers with microfluidic control and single-fluorophore sensitivity. Preliminary data on the CRISPR-Cas9 system showed that a definitive answer to a long-standing question was obtained with rapid turnaround (<2 days) on this ready-to-use, stable, and flexible instrument. Visualization, manipulation and characterization of single molecules are essentially simultaneous, allowing users to correlate mechanical and fluorescence measurements in a controlled and quantitative manner. This unique integration empowers users to study biological processes at the single-molecule level from multiple points of view, providing unparalleled insights into key bio-molecular interactions. We expect that the equipment will have an immediate and lasting impact on a broad range of NIH-funded project areas, including cancer biology, DNA replication and repair, ribosome assembly, genome engineering using CRISPR, translation initiation, transcriptional regulation, and chromatin remodeling and modification.